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Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR). / Hornyák, Ákos; Bálint, Ádám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor.

In: Journal of Virological Methods, Vol. 181, No. 2, 2012, p. 155-163.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

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Hornyák, Ákos; Bálint, Ádám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor / Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).

In: Journal of Virological Methods, Vol. 181, No. 2, 2012, p. 155-163.

Publication: Research - peer-reviewJournal article – Annual report year: 2012

Bibtex

@article{5f4d72bec1264275908ae37411c7399a,
title = "Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)",
keywords = "Feline infectious peritonitis, Sub-genomic messenger RNA, PriProET, Quantitative real-time PCR",
publisher = "Elsevier BV",
author = "Ákos Hornyák and Ádám Bálint and Attila Farsang and Gyula Balka and Mikhayil Hakhverdyan and Rasmussen, {Thomas Bruun} and Jonas Blomberg and Sándor Belák",
year = "2012",
doi = "10.1016/j.jviromet.2012.01.022",
volume = "181",
number = "2",
pages = "155--163",
journal = "Journal of Virological Methods",
issn = "0166-0934",

}

RIS

TY - JOUR

T1 - Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

A1 - Hornyák,Ákos

A1 - Bálint,Ádám

A1 - Farsang,Attila

A1 - Balka,Gyula

A1 - Hakhverdyan,Mikhayil

A1 - Rasmussen,Thomas Bruun

A1 - Blomberg,Jonas

A1 - Belák,Sándor

AU - Hornyák,Ákos

AU - Bálint,Ádám

AU - Farsang,Attila

AU - Balka,Gyula

AU - Hakhverdyan,Mikhayil

AU - Rasmussen,Thomas Bruun

AU - Blomberg,Jonas

AU - Belák,Sándor

PB - Elsevier BV

PY - 2012

Y1 - 2012

N2 - Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10–50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.

AB - Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10–50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.

KW - Feline infectious peritonitis

KW - Sub-genomic messenger RNA

KW - PriProET

KW - Quantitative real-time PCR

U2 - 10.1016/j.jviromet.2012.01.022

DO - 10.1016/j.jviromet.2012.01.022

JO - Journal of Virological Methods

JF - Journal of Virological Methods

SN - 0166-0934

IS - 2

VL - 181

SP - 155

EP - 163

ER -